Apoptosis ; drug effects ; HeLa Cells ; Hepatocytes ; cytology ; drug effects ; Humans ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the effects of X ray on human colorectal cancer cells for their tumor necrosis factor receptor-p55 (TNFR-p55) expression and release of soluble soluble TNFR-p55 (sTNFR-p55) in vitro.

METHODSThe protein expression of TNFR-p55 in Lovo cells exposed to X-ray was detected using immunohistochemistry, and enzyme-linked immunosorbent assay was used to examine the levels of sTNFR-p55 in the supernatants of the cell culture. The cell apoptosis of the exposed cells was analyzed with flow cytometry, and the changes in cell morphology were observed microscopically.

RESULTSX-ray exposure of cells resulted in a strong increase in TNFR-p55 expression of (P<0.01) and LoVo cell apoptosis (P<0.05). The levels of sTNFR-p55 in the supernatant of cells with X-ray exposure was significantly lowered in comparison with the levels before exposure (P<0.01). Optical microscopy showed that the exposed LoVo cells shrank and became spherical with cytoplasmic condensation and nuclear pyknosis.

CONCLUSIONX-ray exposure can induce LoVo cell apoptosis by increasing TNFR-p55 expression on the cell membrane and inhibiting the release of sTNFR-p55 in the supernatants.

Apoptosis ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; metabolism ; Tumor Necrosis Factor Decoy Receptors ; genetics ; metabolism ; X-Rays

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.
Receptors, Tumor Necrosis Factor, Type II/genetics/*metabolism/physiology ; Receptors, Tumor Necrosis Factor, Type I/genetics/*metabolism/physiology ; RNA, Messenger/metabolism ; NF-kappa B/metabolism ; Humans ; Gene Expression Regulation ; Fetus/cytology ; Cytokines/*pharmacology ; Cells, Cultured ; Astrocytes/drug effects/*metabolism

BACKGROUNDRev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE).

METHODSMolecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope.

RESULTSThe new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours.

CONCLUSIONSRev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.

AIDS Vaccines ; immunology ; Apoptosis ; Biolistics ; Cell Line, Tumor ; Gene Products, rev ; physiology ; Genes, env ; physiology ; Genetic Vectors ; Humans ; Keratinocytes ; metabolism ; Plasmids ; Receptors, Tumor Necrosis Factor, Type I ; genetics

BACKGROUNDTumor necrosis factor alpha (TNF-α) is important in promoting relative adrenal insufficiency (RAI) due to systemic inflammatory response syndrome (SIRS). We identified the TNF-α receptor involved in the inhibition of adrenal corticotrophin (ACTH)-stimulated hydrocortisone release by studying the expression of TNF-α receptors in adrenal cortex Y1 cells and the effect of downregulating TNF receptors on ACTH-stimulated hydrocortisone release.

METHODSWe used real-time PCR and immunocytochemistry to evaluate the expression of TNF receptors on Y1 cells. TNF-receptor 1 (TNF-R1) DNA fragments corresponding to the short hairpin RNA (shRNA)-sequences were synthesized and cloned into pcDNA(TM) 6.2-GW/EmGFP expression vector. Knockdown efficiency of TNF-R1 expression was evaluated in miRNA transfected and mock-miRNA transfected Y1 cells by quantitative real-time PCR (Q-PCR). Hydro-cortisone expression levels were determined in TNF-R1-knockdown and control Y1 cells treated with TNF-α and ACTH.

RESULTSMouse adrenal cortex Y1 cells were positive for type I TNF-R1, but not type II TNF-receptor (TNF-R2). Blocking TNF-R1 expression resulted in loss of TNF-α-mediated inhibition of ACTH-stimulated hydrocortisone expression, suggesting a role for the TNF-R1 related signaling pathway in ACTH-stimulated hydrocortisone synthesis.

CONCLUSIONThe inhibitory effect of TNF-α on ACTH-stimulated hydrocortisone synthesis was mediated via TNF-R1 in adrenal cortex.

Adrenal Cortex ; cytology ; drug effects ; metabolism ; Animals ; Cell Line ; Hydrocortisone ; metabolism ; Immunohistochemistry ; Mice ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc (sTNFRI-IgGFc) and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells.

METHODSsTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGloxPE1,3Cre into HEK293 cells. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the transfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry.

RESULTSAd-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 x 10(10) TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the transfected HBE135-E6E7 cells.

CONCLUSIONThe constructed Ad-sTNFRI-IgGFc can effectively infect HBE135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFalpha in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.

Adenoviridae ; genetics ; Bronchi ; cytology ; Epithelial Cells ; cytology ; metabolism ; Genetic Vectors ; genetics ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor, Type I ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo detect the expression of recombinant plasmid PcDNA3.1-sTNFRI in vitro and evaluate the bioactivity of expressed sTNFRI.

METHODSCHO cells were transfected with recombinant plasmid PcDNA3.1-sTNFRI by liposome. sTNFRI in cell culture supernatant was detected by ELISA and sTNFRI blockage of TNF-alpha cytotoxicity in L929 cells evaluated by MTT assay.

RESULTSThe expression of sTNFRI in transfected cell culture supernatant was higher than control groups (P < 0.001). The expressed sTNFRI could significantly neutralize TNF-alpha cytotoxicity in L929 cells.

CONCLUSIONSThese results showed that the recombinant plasmid PcDNA3.1-sTNFRI can be expressed in mammalian cells and the recombinant sTNFRI has biological function.

Animals ; Antigens, Surface ; biosynthesis ; CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; drug effects ; Humans ; Plasmids ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor, Type I ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection

Impaired tumor necrosis factor receptor-1 (TNFR-1) signaling has been found in some malignant tumors with poor prognosis. However, the exact role of TNFR-1 signaling in fibrosarcoma remains unclear. Here, we explored the question by comparing the growth of TNFR-1 deficient (Tnfr1 (-)) and TNFR-1 competent (Tnfr1 (+)) fibrosarcoma FB61 cells (FB61-m and FB61-R1) in mice. TNFR-1 expression on fibrosarcoma cells delayed their growth in vivo but not in vitro. Moreover, reduced FB61-R1 tumor growth was also obtained in TNFR-1 knockout mice. The mechanism relies mainly on the TNFR-1-mediated downregulation of vascular endothelial growth factor (VEGF) production by tumor cells. Importantly, treatment of FB61-m tumors with melphalan resulted in a short delay of tumor growth, followed by a quick remission. However, when FB61-R1 tumors were treated with melphalan, tumor growth was similarly delayed at first and then completely rejected. Our results reveal evidence for TNFR-1 on tumor cells as a prerequisite in chemotherapy for fibrosarcoma, and provide novel insight into the therapeutic approach against some types of tumors using TNFR-1 angonist.
Animals ; Down-Regulation ; drug effects ; Fibrosarcoma ; drug therapy ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Melphalan ; administration & dosage ; Mice ; Molecular Targeted Therapy ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.
Animals ; Chloroquine ; toxicity ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Etanercept ; therapeutic use ; Ganglia, Spinal ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Pruritus ; chemically induced ; drug therapy ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; deficiency ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; deficiency ; genetics ; Signal Transduction ; drug effects ; Skin ; drug effects ; metabolism ; Spinal Cord ; drug effects ; metabolism ; Thalidomide ; therapeutic use ; Time Factors ; Tumor Necrosis Factor-alpha ; adverse effects ; genetics ; metabolism ; p-Methoxy-N-methylphenethylamine ; toxicity

Objective: To summarize the clinical characteristics of tumour necrosis factor receptor-associated periodic syndrome (TRAPS) in children. Methods: The clinical manifestations, laboratory tests, genetic testing and follow-up of 10 children with TRAPS from May 2011 to May 2021 in 6 hospitals in China were retrospectively analyzed. Results: Among the 10 patients with TRAPS, including 8 boys and 2 girls. The age of onset was 2 (1, 5) years, the age of diagnosis was (8±4) years, and the time from onset to diagnosis was 3 (1, 7) years. A total of 7 types of TNFRSF1A gene variants were detected, including 5 paternal variations, 1 maternal variation and 4 de novo variations. Six children had a family history of related diseases. Clinical manifestations included recurrent fever in 10 cases, rash in 4 cases, abdominal pain in 6 cases, joint involvement in 6 cases, periorbital edema in 1 case, and myalgia in 4 cases. Two patients had hematological system involvement. The erythrocyte sedimentation rate and C-reactive protein were significantly increased in 10 cases. All patients were negative for autoantibodies. In the course of treatment, 5 cases were treated with glucocorticoids, 7 cases with immunosuppressants, and 7 cases with biological agents. Conclusions: TRAPS is clinically characterized by recurrent fever accompanied by joint, gastrointestinal, skin, and muscle involvement. Inflammatory markers are elevated, and autoantibodies are mostly negative. Treatment mainly involves glucocorticoids, immunosuppressants, and biological agents.
Male ; Child ; Female ; Humans ; Child, Preschool ; Receptors, Tumor Necrosis Factor, Type I/genetics* ; Retrospective Studies ; Hereditary Autoinflammatory Diseases/drug therapy* ; Glucocorticoids/therapeutic use* ; Biological Factors/therapeutic use* ; Immunosuppressive Agents/therapeutic use* ; Autoantibodies ; Familial Mediterranean Fever/diagnosis* ; Mutation